XopJ suppresses cell death during the compatible Xcv-pepper interaction.

(A) Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA), XopJ (G2A-HA) and (C235A-HA) were inoculated at a bacterial density of 2×108 cfu ml−1 into leaves of pepper ECW plants. Leaf phenotype was photographed at 3 d post infection (dpi). (B) Protein extracts from pepper leaves infiltrated with 1 mM MgCl2, Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA), XopJ (G2A-HA) and (C235A-HA) at 3 dpi were prepared. Equal volumes representing approximately equal protein amounts of each extract were immunoblotted and proteins were detected using anti-HA antiserum. (C) Trypan blue staining of infected tissue of ECW plants reveals reduced cell death in the presence of functional XopJ. Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA), XopJ (G2A-HA) and (C235A-HA) were inoculated at a bacterial density of 2×108 cfu ml−1 into leaves of pepper ECW plants. Samples of infected and untreated leaves were taken 3 dpi and stained with trypan blue. Dead plant cells stain blue. Grey and black spots represent calcium oxalate crystals. (D) Summary of observed phenotypes. 20 pepper plants were infected with the different Xcv strains indicated in this figure and then scored for phenotype development at 3 dpi. (E) Delivery of XopJ by Xcv leads to reduced ion leakage in pepper. Ion leakage was measured in pepper plants infected with. Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA), XopJ (G2A-HA) and (C235A-HA). Conductivity was measured at the time points indicated. Data represent the mean SD (n = 3). Significant differences are indicated by asterisks (* P(A) 将携带空载体的野油菜黄单胞菌辣椒致病变种（Xcv）、ΔxopJ基因缺失突变体（Xcv ΔxopJ，携带空载体）、ΔxopJ基因缺失互补菌株（Xcv ΔxopJ (XopJ-HA)，即表达XopJ与血凝素（hemagglutinin, HA）标签融合蛋白的菌株）、XopJ(G2A-HA)突变体菌株及XopJ(C235A-HA)突变体菌株，以2×10⁸菌落形成单位每毫升（cfu ml⁻¹）的浓度接种至辣椒ECW品种植株叶片中，于接种后3天（days post infection, dpi）拍摄叶片表型。 (B) 于接种后3天，提取经1 mM氯化镁（MgCl₂）缓冲液、携带空载体的Xcv、Xcv ΔxopJ（空载体）、Xcv ΔxopJ (XopJ-HA)、XopJ(G2A-HA)及XopJ(C235A-HA)菌株真空渗透处理的辣椒ECW品种叶片总蛋白。取等体积、蛋白含量大致相当的各蛋白提取物进行免疫印迹实验，采用抗HA抗血清检测目标蛋白。 (C) 台盼蓝（Trypan blue）染色结果显示，功能型XopJ的存在可减弱ECW植株的细胞死亡程度。将上述菌株以2×10⁸ cfu ml⁻¹的浓度接种至辣椒ECW品种植株叶片，于接种后3天采集接种及未接种的叶片样本进行台盼蓝染色：死亡的植物细胞会被染为蓝色，灰黑色斑点则为草酸钙结晶。 (D) 本研究选取20株辣椒ECW品种植株，分别接种本图所示的不同Xcv菌株，于接种后3天统计并总结观测到的表型变化。 (E) 野油菜黄单胞菌辣椒致病变种递送的XopJ可降低辣椒叶片的离子渗漏。分别以携带空载体的Xcv、Xcv ΔxopJ（空载体）、Xcv ΔxopJ (XopJ-HA)、XopJ(G2A-HA)及XopJ(C235A-HA)菌株接种辣椒ECW品种植株，按指定时间点检测电导率。数据以平均值±标准差（standard deviation, SD）表示（n = 3），显著性差异以星号标注（* P